Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS

Abstract

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40–100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.

The Disulfide Bond, but Not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu,Zn Superoxide Dismutase (SOD1) Fibrillation

“…Both wild-type and ALS-mutant human SOD1 can be induced to aggregate in vitro using a variety of different treatments, including low concentrations of reducing agents such as DTT or glutathione (16 -19), low pH (18,20), structural perturbants such as trifluoroethanol and elevated temperature (21,22), oxidative modification induced by hydrogen peroxide (23), and by long term air exposure leading to cross-linking of nondisulfide cysteines (24). Some of these conditions, notably low pH, elevated temperatures, trifluoroethanol, and incubation in reducing agents, generate aggregates that bear a remarkable resemblance to amyloid fibrils.…”

Identification of a novel Cys146X mutation of SOD1 in familial amyotrophic lateral sclerosis by whole-exome sequencing

“…19 However, variable clinical features and courses, due to the loss of disulfide bonds of SOD1, 20 suggest that other factors may be involved in the pathogenesis of the disease.…”

Folding and Aggregation of Cu, Zn-Superoxide Dismutase

“…A number of studies have used different solution conditions (increased temperature, decreased pH, increased ionic strength, sonication or agitation) to promote the formation of well-structured, fibrillar amyloid aggregates (see 1.2.3) by various forms of SOD1 (Stathopulos et al 2004;Chattopadhyay et al 2008;Chattopadhyay and Valentine 2009;Oztug Durer et al 2009). Other studies have demonstrated soluble oligomer and small aggregate formation by various forms of SOD1 in quiescent, physiologically relevant solution conditions (Vassall, 2011, Hwang, 2010, Banci, 2008. Thus, it is evident that multiple factors can greatly influence protein folding and aggregation and these factors must be considered when investigating the molecular mechanisms of protein aggregation.…”